Requirement for high mobility group protein HMGI-C interaction with STAT3 inhibitor PIAS3 in repression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) transcription by Ras activation in salivary epithelial cells.

Previously, we have demonstrated that oxidative stress or Ras/ERK activation leads to the transcriptional repression of alpha-subunit of epithelial Na(+) channel (ENaC) in lung and salivary epithelial cells. Here, we further investigated the coordinated molecular mechanisms by which alpha-ENaC expression is regulated. Using both stable and transient transfection assays, we demonstrate that the overexpression of high mobility group protein I-C (HMGI-C), a Ras/ERK-inducible HMG-I family member, represses glucocorticoid receptor (GR)/dexamethasone (Dex)-stimulated alpha-ENaC/reporter activity in salivary epithelial cells. Northern analyses further confirm that the expression of endogenous alpha-ENaC gene in salivary Pa-4 cells is suppressed by an ectopic HMGI-C overexpression. Through yeast two-hybrid screening and co-immunoprecipitation assays from eukaryotic cells, we also discovered the interaction between HMGI-C and PIAS3 (protein inhibitor of activated STAT3 (signal transducer and activator of transcription 3)). A low level of ectopically expressed PIAS3 cooperatively inhibits GR/Dex-dependent alpha-ENaC transcription in the presence of HMGI-C. Reciprocally, HMGI-C expression also coordinately enhances PIAS3-mediated repression of STAT3-dependent transactivation. Moreover, overexpression of antisense HMGI-C construct is capable of reversing the repression mediated by Ras V12 on GR- and STAT3-dependent transcriptional activation. Together, our results demonstrate that Ras/ERK-mediated induction of HMGI-C is required to effectively repress GR/Dex-stimulated transcription of alpha-ENaC gene and STAT3-mediated transactivation. These findings delineate a network of inhibitory signaling pathways that converge on HMGI-C.PIAS3 complex, causally associating Ras/ERK activation with the repression of both GR and STAT3 signaling pathways in salivary epithelial cells.

Oxidative stress disrupts glucocorticoid hormone-dependent transcription of the amiloride-sensitive epithelial sodium channel alpha-subunit in lung epithelial cells through ERK-dependent and thioredoxin-sensitive pathways.

The amiloride-sensitive epithelial Na(+) channel (ENaC) plays a critical role in the maintenance of alveolar fluid balance. It is generally accepted that reactive oxygen and nitrogen species can inhibit ENaC activity and aggravate acute lung injury; however, the molecular mechanism for free radical-mediated ENaC inhibition is unclear. Previously, we showed that the expression of the alpha-subunit of ENaC, alpha-ENaC, which is indispensable for ENaC activity, is repressed by Ras activation in salivary epithelial cells. Here, we investigated whether exogenous H(2)O(2) modulates alpha-ENaC gene expression in lung epithelial cells through a similar molecular mechanism. Utilizing transient transfection reporter assays and site-directed mutagenesis analyses, we found that the glucocorticoid response element (GRE), located at -1334 to -1306 base pairs of the alpha-ENaC 5'-flanking region, is the major enhancer for the stimulated alpha-ENaC expression in A549 lung epithelial cells. We further demonstrate that the presence of an intact GRE is necessary and sufficient for oxidants to repress alpha-ENaC expression. Consistent with our hypothesis, exogenous H(2)O(2)-mediated repression of alpha-ENaC GRE activity is partially blocked by either a specific inhibitor for extracellular signal-regulated kinase (ERK) pathway activation, U0126, or dominant negative ERK, suggesting that, in part, activated ERK may mediate the repressive effects of H(2)O(2) on alpha-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the alpha-ENaC GRE in the presence of exogenous H(2)O(2). Taken together, we hypothesize that oxidative stress impairs Na(+) transport activity by inhibiting dexamethasone-dependent alpha-ENaC GRE activation via both ERK-dependent and thioredoxin-sensitive pathways. These results suggest a putative mechanism whereby cellular redox potentials modulate the glucocorticoid receptor/dexamethasone effect on alpha-ENaC expression in lung and other tight epithelia.

The gene expression of the amiloride-sensitive epithelial sodium channel alpha-subunit is regulated by antagonistic effects between glucocorticoid hormone and ras pathways in salivary epithelial cells.

The functional expression of the amiloride-sensitive epithelial sodium channel (ENaC) in select epithelia is critical for maintaining electrolyte and fluid homeostasis. Although ENaC activity is strictly dependent upon its alpha-subunit expression, little is known about the molecular mechanisms by which cells modulate alpha-ENaC gene expression. Previously, we have shown that salivary alpha-ENaC expression is transcriptionally repressed by the activation of Raf/extracellular signal-regulated protein kinase pathway. Here, this work further investigates the molecular mechanism(s) by which alpha-ENaC expression is regulated in salivary epithelial Pa-4 cells. A region located between -1.5 and -1.0 kilobase pairs of the alpha-ENaC 5'-flanking region is demonstrated to be indispensable for the maximal and Ras-repressible reporter expression. Deletional analyses using heterologous promoter constructs reveal that a DNA sequence between -1355 and -1269 base pairs functions as an enhancer conferring the high level of expression on reporter constructs, and this induction effect is inhibited by Ras pathway activation. Mutational analyses indicate that full induction and Ras-mediated repression require a glucocorticoid response element (GRE) located between -1323 and -1309 base pairs. The identified alpha-ENaC GRE encompassing sequence (-1334/-1306) is sufficient to confer glucocorticoid receptor/dexamethasone-dependent and Ras-repressible expression on both heterologous and homologous promoters. This report demon- strates for the first time that the cross-talk between glucocorticoid receptor and Ras/extracellular signal-regulated protein kinase signaling pathways results in an antagonistic effect at the transcriptional level to modulate alpha-ENaC expression through the identified GRE. In summary, this study presents a mechanism by which alpha-ENaC expression is regulated in salivary epithelial cells.

Infant temperament and anxious symptoms in school age children.

A group of 164 children from different infant temperament categories were seen at 7 years of age for a laboratory battery that included behavioral and physiological measurements. The major results indicated that children who had been classified as high reactive infants at 4 months of age, compared with infants classified as low reactive, (a) were more vulnerable to the development of anxious symptoms at age 7 years, (b) were more subdued in their interactions with a female examiner, (c) made fewer errors on a task requiring inhibition of a reflex, and (d) were more reflective. Further, the high reactives who developed anxious symptoms differed from the high reactives without anxious symptoms with respect to fearful behavior in the second year and, at age 7 years, higher diastolic blood pressure, a narrower facial skeleton, and greater magnitude of cooling of the temperature of the fingertips to cognitive challenge. Finally, variation in magnitude of interference to fearful or aggressive pictures on a modified Stroop procedure failed to differentiate anxious from nonanxious or high from low reactive children.

The amiloride-sensitive epithelial sodium channel alpha-subunit is transcriptionally down-regulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway.

Previous studies have shown that an inducible Raf-1 kinase protein, DeltaRaf-1:ER, activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell line, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by DeltaRaf-1:ER activation. Through this approach, the gene encoding the alpha-subunit of the amiloride-sensitive epithelial sodium channel (alpha-ENaC) was identified as a target of activated Raf-1 kinases. alpha-ENaC down-regulation could also be seen in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA), indicating that the repression of steady-state alpha-ENaC mRNA level was dependent upon the activity of protein kinase C, the target of TPA, as well. Pretreatment of cells with a specific inhibitor of the ERK kinase pathway, PD 98059, markedly abolished the down-regulation of alpha-ENaC expression, consistent with the hypothesis that the ERK kinase-signaling pathway is involved in TPA-mediated repression. Moreover, through the use of transient transfection assays with alpha-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the alpha-subunit may provide new insights into the modulation of sodium reabsorption across epithelia.

Early childhood predictors of adult psychopathology.

This paper considers the relationship between experience and behavioral profiles during the first 4 years of life and later psychopathology by examining the results of prospective longitudinal studies. The review revealed modest associations. The most consistent findings imply that an extreme degree of impulsivity in preschool children predicts adolescent delinquency, and that severe neuromotor anomalies in infants are a possible sign of vulnerability to adult schizophrenia.

Identification of a region important for human monoamine oxidase B substrate and inhibitor selectivity.

Monoamine oxidase (MAO) A and B are flavoenzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. To search for domains that confer substrate and inhibitor selectivities, two chimeric proteins were constructed and expressed in yeast. The kinetic constants and IC50 values were determined for these chimeric enzymes using MAO-A/B selective substrates and inhibitors. Replacement of MAO-A amino acids 161-375 with the corresponding region of MAO-B, termed AB(161-375)A, converted MAO-A catalytic properties to MAO-B like ones. The specificity constants (k(cat)/K(m))for the oxidation of beta-phenylethylamine (PEA) (1.6 x 10(5) s-1 M-1) and benzylamine (2.4 x 10(4) s-1 M-1) by AB (161-375)A were similar to wild-type MAO-B (PEA, 8 x 10(5)s(-1) M(-1); benzylamine, 4.9 x 10(4) s(-1) M(-1). Serotonin (5-HT), a preferred substrate for MAO-A, was not oxidized by AB(161-375)A or wild-type MAO-B. Furthermore, (AB161-375)A was more sensitive to the MAO-B specific inhibitor deprenyl (IC50 2.7 +/- 0.4 x 10(-8) M) than to the MAO-A specific inhibitor clorgyline (IC50 5.4 +/- 0.8 x 10(-7) M). However, the reciprocal chimera in which a MAO-B segment was replaced with the corresponding region of MAO-A, termed ++(+BA152-366B), lacked catalytic activity. The lack of catalytic activity was not due to aberrant expression but rather an inactive protein as demonstrated by Western blot analysis. These results demonstrate that MAO-B amino acids 152-366 contain a domain(s) that confers substrate and inhibitor selectivity.

A fetal alcohol syndrome surveillance pilot project in American Indian communities in the Northern Plains.

A pilot fetal alcohol syndrome (FAS) surveillance was carried out in four American Indian communities in the Northern Plains by the Aberdeen Area Indian Health Service to determine the incidence of FAS and to evaluate the feasibility of establishing continuing surveillance for FAS. Baseline data on the incidence of FAS would be used by the Indian Health Service to develop and evaluate preventive interventions, including treatment programs for pregnant women who drink alcohol. Four of the 1,022 children included in the project were found to have FAS, a rate of 3.9 per 1,000 live births. The rate is believed to underestimate the true rate of FAS because some low birth weight children were not screened, parents or guardians were reluctant to bring children suspected of FAS for evaluation, clinicians were hesitant to diagnose possible alcohol-damaged children for fear of labeling the child, and some children with FAS died before the diagnosis of FAS could be confirmed. If the rate of FAS is similar for the 39 percent of the infants not screened and for the 25 percent of suspected infants who were not evaluated, a rate of 8.5 cases of FAS per 1,000 live births may be postulated. The authors recommend routine screening of prenatal patients for substance abuse and establishing a tracking system for low birth weight infants suspected to have FAS or other alcohol-related developmental disorders, in an effort to establish more accurate FAS rates. Such a surveillance system would identify women at risk of having alcohol-affected infants so that appropriate treatment and counseling could be provided, possibly reducing the severity of adverse effects of alcohol on their fetuses.

[Temperament, psychological development and psychopathology. Correlations, explanatory models and forms of intervention]. / Temperament, psychische Entwicklung und Psychopathologie. Zusammenhänge, Erklärungsmodelle und Formen der Intervention.

A return to temperamental concepts characterizes contemporary child psychology in the United States. The word "temperament" refers to early developing differences in reactivity and behavioral style. Temperament research focuses on the origins, the stability, the dimensionality and the psychopathological significance of such differences. This article especially emphasizes the latter. Associations between temperament and psychopathology are noted. Chess' and Thomas' goodness of fit-model is presented as an explanatory model for these associations. According to this model, behavior disorders result from an incompatibility between the normal variations of child and environment. The applicability of this model in clinical and educational settings is then outlined. Finally, the author considers the contribution of temperament research to established paradigms in developmental psychopathology.